Inflammatory Pain - Control of Immune Cell Nicotinic Receptor Signaling

Signaling from the nervous system to peripheral organs via the sympathetic and parasympathetic nervous system has been well documented for the autonomic control of inner organs, e.g. in the cardiovascular or gastro-intestinal system. In particular, the cholinergic system uses the neurotransmitter acetylcholine that acts on muscarinic receptors in the autonomic nervous system but also on nicotinic receptors (nAChR) at the neuromuscular junction, central nervous system neurons and autonomic ganglia. Nicotinic receptors act as ionotropic, ligand-gated ion channels that are composed of 1-5 distinct subunits building a functional pentamer. Currently, ten a (a1-10), four ß (ß1-4), one g, d, and e nAChRs have been identified through molecular cloning, and besides the classical target organs, intriguingly, leukocytes of the immune system (e.g. macrophages, B-cells, T-cells, monocytes) have also been found to express nAChRs, e.g. homomeric a7 nAChR (Fuji et al. 2017, J Pharmacol Sci 4). For example, smoking, associated with nicotine exposure to the blood, has been found to activate a7 nAChR and in turn, to reduce LPS-induced pro-inflammatory TNF-a release (Fuji et al. 2017). Thus, nicotinic signaling clearly modulates the immune response. On the other hand, a7 nAChRs are also involved in mediating chronic neuropathic and inflammatory pain (Alsharari et al. 2013, Biochem Pharmacol). Conotoxins are a class of peptide-based drugs derived from cone snail venom and that were shown to be potent modulators of chronic pain via acting on a9-containing nAChRs (Hone et al. 2017, Br J Pharmacol). Since the Australian partner has a long-standing experience working with conotoxins, our project hypothesis is that conotoxins may be also suitable drug leads to modulate inflammatory pain by specific action of immune modulation in leukocytes. Thus, an important information still missing in the literature is represented by the question of how conotoxins may act on nAChRs composed of various isoforms usually found in immune cells to down-modulate immune activation, immunological hyper-reactivity and eventually, inflammatory pain. We thus propose a combined high content-high resolution screening platform from previous work to be applied to the studies of ACh-activated Ca²⁺ signaling in various human immune cell types and its inhibition by conotoxins as potential compounds to address inflammatory pain.

The specific goals of this project are:

1)      To use selective antagonists a-conotoxins potentially directed against the most common isoforms (a7, a9, a10) against nAChRs in human immune cell lines (U937 monocytic cells, Jurkat T-cells, RPMI-A226 B-cells) to monitor intracellular Ca²⁺ responses using Fura-2 monitoring of nAChR responses in plate reader assays. Concentration-response relationships will be established.

2)      To determine the most potent concentrations to apply conotoxins in high resolution single cell Ca²⁺ imaging studies in the aforementioned cell lines to determine mechanisms of conotoxin-induced modulation of Ca²⁺-signaling.

3)      To perform nAChR isoform expression profiling and comparing results from stable immune cell lines with heterologous expression systems varying isoform composition to obtain a pharmaco-biophysical profiling.